1. Field of Invention
The present invention relates to a method for culturing microorganisms having a methanol metabolic pathway into which has been introduced an expression unit in which a target gene is linked downstream to a promoter able to be induced by methanol, said method enabling the target gene product to be produced efficiently.
2. Related Art
The production of polypeptides and other gene products using microorganisms as hosts has basically become possible due to the advances made in recombinant DNA technology. Polypeptide production is performed by introducing an expression unit in which the gene for a target polypeptide is linked downstream to a suitable promoter. Microorganisms such as E. coli, yeasts and animal cells are typically used as a host.
Gene expression systems using E. coli as the host are the most commonly used systems, and these systems typically provide a high productivity. However, since there are many cases in which the expressed polypeptide forms insoluble inclusion bodies resulting in an insoluble form, the possibility of using this system is largely dependent on the properties of the target polypeptide.
On the other hand, gene expression systems that use animal cells as a host are useful for the purpose of confirming an activity of a gene product and so forth since in many cases the expressed polypeptide retains activity. However, the amount of target polypeptide is typically low, and considerable efforts are required for purification in this case. In addition, since animal cells reproduce slowly and the media used are expensive, culturing requires both considerable time and cost. Moreover, it is also difficult to increase the scale of culturing. Consequently, animal cells are not desirable as a host for industrially obtaining a target polypeptide in large volume.
Yeast cells are eucaryotic cells having endoplasmic reticulum, Golgi apparatus and other cellular components similar to animals cells. They are also known to be able to form and express polypeptides having particularly tertiary structures in polypeptides derived from eucaryotic cells. In addition, they reproduce faster than animal cells and can easily be cultured in large volume. Genetic analyses have been conducted most extensively on Saccharomyces cerevisiae, and it is widely used at the laboratory level as a host for gene expression.
However, since it is difficult to grow Saccharomyces cerevisiae to a high cell density and yield per culture medium is not high, it is not sufficient for industrial production of the target polypeptide. In order to solve this problem, methylotrophic yeasts such as Pichia pastoris, Hansenula polymorpha and Candida boidinii are used.
For example, Gellissen et al. succeeded in producing 1.4 g of glucoamylase at a cell density of 100 to 130 g dry cell weight per liter of culture medium using a formic dehydrogenase promoter for which expression is induced by methanol and Hansenula polymorpha as host (Gellissen et al. BIO/TECHNOLOGY, 9, 291-295, 1991). In addition, Barr et al. succeeded in producing 4 g of human serum albumin per liter of culture medium using an alcohol oxidase promoter for which expression is induced by methanol and Pichia pastoris as host (Barr et al. Pharm. Eng., 12(2), 48-51, 1992).
However, the problems described below are still possible even in the case of methods using methylotrophic yeasts. In the case of a yeast having a methanol metabolic pathway in a heterogeneous gene expression system using a yeast represented by the above-mentioned three species of methylotrophic yeasts and a promoter able to be induced by methanol, the methanol in the culture medium rapidly decreases when the yeast is cultured in a medium containing methanol. In this type of culturing, methanol must be supplied as a carbon source in order to simultaneously maintain transcription from the promoter and cell growth.
However, if the methanol concentration in the culture medium is suddenly increased during supply of methanol, the yeast may be killed. In addition, measurement of the methanol concentration in a culture of yeast having a methanol metabolic pathway is generally performed by applying the supernatant to gas chromatography and so forth. In this method, however, in addition to requiring special equipment, since a considerable amount of time is required until methanol concentration can be determined, it has the disadvantage of preventing a rapid judgment from being made regarding the need for replenishment of methanol.
As an example for a method for maintaining methanol concentration in a culture medium for expression of a target gene, a method is used that reduces the methanol consumption rate using a yeast in which the enzyme alcohol oxidase is missing from its metabolic pathway. This method involves growing yeast using glycerol and so forth as a carbon source, and then inducing transcription from a promoter by adding a fixed amount of methanol to the culture medium to express the target gene. Due to the low rate of methanol consumption, it is easy to maintain methanol concentration. Although it is possible to obtain a stable culture, this method has the disadvantage of requiring a long culturing time since culturing is performed separately for a cell growth period and a target gene expression period.
A method is disclosed in WO 95/21928 wherein the methanol concentration in a culture medium is controlled to a constant level in order to express a target gene by culturing a methylotrophic yeast, in which methanol metabolism has been slowed by partially altering enzymes of metabolic pathway, without increasing the number of cells. This method involves measuring a methanol concentration in the air inside the culture tank in a state in which a methanol concentration in the air inside the culture tank reflects the methanol concentration in the culture medium, and then determining the methanol addition rate from those results to control the methanol concentration in the culture medium.
The methanol concentration in a culture medium of methylotrophic yeast deficient for alcohol oxidase gene can be controlled to a constant level by applying this method. However, in order to predict the methanol concentration in the culture medium from the methanol concentration in the air inside the culture tank, it is necessary that both be at equilibrium. In order to achieve this equilibrium, culture conditions including aeration rate, pressure and temperature must be maintained constant, and the methanol concentration in the culture medium must not change suddenly. Due to these restrictions, the above-mentioned method for controlling methanol concentration could only be applied to culturing in the gene expression induction phase after the cells had been grown in advance using a yeast that does not have a methanol metabolic pathway.
On the other hand, in a heterogeneous gene expression system using a promoter capable of being induced with methanol and a microorganism having a methanol metabolic pathway as a host, it is not necessary to isolate the microorganism growth phase and the induction phase for gene expression if the rate of methanol addition and the concentration of methanol in the culture medium are suitably controlled. Consequently, culturing time is shortened which results in the target protein being able to be obtained in a short time. Thus, in the culturing of microorganisms having a methanol metabolic pathway, there is a need to establish a method for maintaining a suitable rate of methanol addition or methanol concentration that satisfies both the production of the target gene product and microorganism growth.